RCK Domain Research Articles

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

Cysteine residue 911 in C-terminal tail of human BKCaα channel subunit is crucial for its activation by carbon monoxide

The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).

On the Properties of the RCK1 Domain of the Human BK (SLO1) Channel

In BK channels, four RCK1 and four RCK2 domains assemble into a cytosolic ligand-sensing superstructure known as the gating ring. While electrophysiological data suggest that both RCK1 and RCK2 contain high affinity Ca2+-sensing sites, recent crystallographic data (Wu et al., 2010) has revealed Ca2+ binding only within the RCK2 domain. Does the RCK1 domain bind Ca2+? What is the role of the high-affinity Ca2+ sensing residues D362/D367 (Xia, et al 2002) located in RCK1 domain? To address these questions, we expressed and purified the region of the human BK channel C-terminus corresponding to the amino acid sequence 322IIE¼HDP667. We have probed the structure and Ca2+ sensing properties of the RCK1 domain in solution, under physiologically relevant conditions. In addition to the α/β fold shared with its bacterial counterparts and human BK RCK2 domain, the RCK1 domain preferentially self-assembles into a homo-octameric structure. We recently reported that RCK1 undergoes Ca2+-dependent conformational changes similarly to the RCK2 domain (Yusifov et al., 2008 and 2010). The neutralization of residues D362/D367 altered the secondary and quaternary structure of the RCK1 domain and prevented Ca2+-induced structural transitions in RCK1. 45Ca2+ overlay assay suggested that the neutralization of D362/367 did not abolish the Ca2+-binding activity of RCK1 in the range of 2.1-115 µM free Ca2+. While it cannot be excluded that D362/D367 are elements of a Ca2+ binding site with multiple coordinating residues, our results favor the view that D362 /D367 have a predominantly structural role. Possibly, these mutations set RCK1 domains in a conformational state that hampers the propagation of structural changes caused by ligand binding.

Ca 2+ Dependent Activation of Large Conductance Ca 2+ Activated Potassium (BK) Channels by Binding to the RCK1 Domain

BK channels sense intracellular Ca2+ and are important modulators of muscle contraction, neuronal spike frequency adaptation, neurotransmitter release and circadian pacemaker output. The cytosolic domain (CTD) of BK channels contains two structural sub-domains, RCK1 and RCK2. Mutagenesis studies have identified a series of Asp residues in the RCK2 domain (termed as the Ca2+ bowl) and Asp367 in the RCK1 domain as two putative Ca2+ binding sites. A recently published crystal structure of the BK CTD showed Ca2+ binding to the Ca2+ bowl but, surprisingly, provided less further information about the Ca2+ binding site in RCK1. We have performed mutational scan in the RCK1 domain to search for the residues that are necessary for Ca2+ binding in addition to Asp367. Here we show that the mutations of Glu535 in the RCK1 domain produce nearly identical functional consequences on the Ca2+ dependent activation as the mutations of Asp367. Therefore, Glu535, same as Asp367, may be one of binding coordinators for Ca2+ binding in RCK1. We also show that mutations of Met513, some of which have been previously shown to reduce Ca2+ sensitivity, result in a different pattern of functional consequences than those of Glu535 and Asp367, which suggest that Met513 is not part of the Ca2+ binding site. Molecular modeling and experimental data suggest that the binding of Ca2+ by the side chains of Glu535 and Asp367 changes the conformation around the binding site and turns the side chain of Met513 into a hydrophobic core, thereby opening the activation gate of BK channels. We have also investigated Cd2+ dependent activation of BK channels and found that Ca2+ and Cd2+ interact with different sets of residues to activate BK channels.

Investigating Structural Re-Arrangement of the BK Channel Rck1-Rck2 Linker Using Fluorescence Lifetime Imaging Microscopy

Alternative splicing generates considerable functional diversity of large conductance calcium- and voltage-activated potassium (BK) channels. For example, inclusion of the STREX (STRess-activated EXon) insert into the unstructured cytosolic linker between the two RCK domains of the channel generates a cysteine rich domain (CRD) that confers intrinsic hypoxia sensitivity to the channel [1]. Here, we have developed YFP-mCFP fluorescent fusion proteins of the CRD and exploit these to examine conformational rearrangements in the CRD in response to hypoxia using fluorescence lifetime imaging microscopy (FLIM). The fusion protein, expressed in HEK293 cells, is targeted to the plasma membrane and responds to acute hypoxia (10 minute exposure at <5% oxygen) with a significant shift in fluorescence lifetime distribution (n=7). Pre-treatment with either oxidizing or reducing agents did not block the hypoxia induced shift in fluorescence lifetimes (n=4). In contrast, site directed mutagenesis of the cysteine residues within STREX, which we had previously shown to confer functional hypoxia sensitivity to the channel, abolished the hypoxia induced shift in lifetimes (n=6). The effects of cysteine mutagenesis and REDOX manipulation on hypoxia sensitivity were recapitulated in patch clamp assays of channel activity supporting a role for hypoxia-induced shifts in CRD conformation with changes in channel function. These findings suggest that FLIM, in conjunction with patch clamp electrophysiology, will be an important approach to monitor conformational rearrangements and investigate the structure-function relationship of the important unstructured linker region between the channel RCK domains. [1] McCartney, C.E., McClaffert, H., Huibant, J.M., Rowan, E.G., Shipston, M.J, and Rowe. I.C. (2005) A cysteine-rich motif confers hypoxia sensitivity to mammalian large conductance voltage- and Ca-activated K (BK) channel alpha-subunits. Proc. Natl. Acad. Sci. USA 102:17870-17876

C. Elegans Slo-2b uses its RCK1 Domain as a Ca 2+ Sensor and does not Exhibit Cl - Dependence

Slo-2 channels play an important role in the adaption of neuronal firing rates and have been implicated in protection against ischemia. Slo-2 channels belong to the family of high-conductance potassium channels but their gating mechanism is unique and has been reported to exhibit species differences. The rat Slo2 (Slack) channel is activated by Na+ and Cl-, whereas the C.elegans Slo-2a has been reported to be sensitive to Ca2+ and Cl-. Here, we report isolation of a novel isoform of the C. elegans channel Slo-2b (F08b12.3c) that was cloned from ESTs (YK1522e1, YK1193) of C.elegans, which has a distinct N-terminal region (by 18 amino acids) compared to the previously reported Slo-2a (F08b12.3b) (Yuan et al, 2000).

Calcium-Dependent Operation of the Human BK Channel Gating Ring Apparatus

The large cytoplasmic C-terminal domain (CTD) of the human BK channel forms a gating ring structure composed by two tandem RCK domains serving as a signal transducer for intracellular Ca2+ and other ligands. However, the mechanism of the gating ring's operation remains unknown. We have used steady-state and time-resolved spectroscopy in combination with dynamic light scattering to the Ca-induced conformational changes of the purified CTD of human BK channel. The CTD domain of the human BK channel assembles as a tetrameric gating ring structure (MW∼310 kDa) with a hydrodynamic radius (RH) ∼10.5 nm. In the presence of 35 μM free Ca2+, RH reversibly decreases to ∼7.5 nm. The modulation of the gating ring hydrodynamic shape suggests that it undergoes Ca2+-induced conformational transitions, which we further, characterized using time-correlated single-photon counting spectroscopy. Increasing free [Ca2+] up to 35 μM shortened the average Trp fluorescence lifetime (τavg) of the wild-type gating ring from ∼2.6ns to ∼1.9ns, while the neutralization of the high-affinity Ca-binding site within RCK2 (Ca bowl, D894-898N) attenuated the effect of Ca2+. Steady-state fluorescence analysis revealed that these ligand-dependent structural rearrangements of the gating ring possess strong divalent cation selectivity. The gating ring exhibited i) high-affinity Ca2+-binding (Khalf_1 ∼0.3μM and Khalf_2 ∼4μM); ii) significantly lowered affinity for Mg2+ (Khalf ∼200μM) and iii) no Ba2+ sensitivity up to 13mM, consistent with the lack of Ba2+-dependent BK channel activation. Interestingly, Ca2+ bowl neutralization eliminated Mg2+ sensing up to 12mM. In summary, under physiologically-relevant conditions, these ligand-induced conformational transitions are strongly ion-specific and associated with changes in the hydrodynamic properties of the BK gating ring and likely represent the ligand-induced molecular events underlying channel activation.

Modulation of BK Channel Gating by the β2 Subunit Involves Both Membrane-Spanning and Cytoplasmic Domains of Slo1

Large-conductance, Ca(2+)- and voltage-sensitive K(+) (BK) channels regulate neuronal functions such as spike frequency adaptation and transmitter release. BK channels are composed of four Slo1 subunits, which contain the voltage-sensing and pore-gate domains in the membrane and Ca(2+) binding sites in the cytoplasmic domain, and accessory β subunits. Four types of BK channel β subunits (β1-β4) show differential tissue distribution and unique functional modulation, resulting in diverse phenotypes of BK channels. Previous studies show that both the β1 and β2 subunits increase Ca(2+) sensitivity, but different mechanisms may underline these modulations. However, the structural domains in Slo1 that are critical for Ca(2+)-dependent activation and targeted by these β subunits are not known. Here, we report that the N termini of both the transmembrane (including S0) and cytoplasmic domains of Slo1 are critical for β2 modulation based on the study of differential effects of the β2 subunit on two orthologs, mouse Slo1 and Drosophila Slo1. The N terminus of the cytoplasmic domain of Slo1, including the AC region (βA-αC) of the RCK1 (regulator of K(+) conductance) domain and the peptide linking it to S6, both of which have been shown previously to mediate the coupling between Ca(2+) binding and channel opening, is specifically required for the β2 but not for the β1 modulation. These results suggest that the β2 subunit modulates the coupling between Ca(2+) binding and channel opening, and, although sharing structural homology, the BK channel β subunits interact with structural domains in the Slo1 subunit differently to enhance channel activity.

Ion sensing in the RCK1 domain of BK channels

BK-type K(+) channels are activated by voltage and intracellular Ca(2+), which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different Ca(2+) binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this Ca(2+) binding site. This site is specific for binding to Ca(2+) but not Cd(2+). Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of Ca(2+) by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how Ca(2+) binding at this site opens the activation gate of the channel that is remotely located in the membrane.

α-Catulin CTN-1 is required for BK channel subcellular localization in C. elegans body-wall muscle cells

The BK channel, a voltage- and Ca(2+)-gated large-conductance potassium channel with many important functions, is often localized at specific subcellular domains. Although proper subcellular localization is likely a prerequisite for the channel to perform its physiological functions, little is known about the molecular basis of localization. Here, we show that CTN-1, a homologue of mammalian α-catulin, is required for subcellular localization of SLO-1, the Caenorhabditis elegans BK channel α-subunit, in body-wall muscle cells. CTN-1 was identified in a genetic screen for mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of SLO-1. In body-wall muscle cells, CTN-1 coclusters with SLO-1 at regions of dense bodies, which are Z-disk analogs of mammalian skeletal muscle. In ctn-1 loss-of-function (lf) mutants, SLO-1 was mislocalized in body-wall muscle but its transcription and protein level were unchanged. Targeted rescue of ctn-1(lf) in muscle was sufficient to reinstate the lethargic phenotype in slo-1(gf);ctn-1(lf). These results suggest that CTN-1 plays an important role in BK channel function by mediating channel subcellular localization.

The RCK1 domain of the human BKCa channel transduces Ca2+ binding into structural rearrangements

Large-conductance voltage- and Ca2+-activated K+ (BKCa) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca2+ homeostasis. The molecular mechanism of Ca2+-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BKCa RCK1 domain adopts an α/β fold, binds Ca2+, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca2+-induced conformational changes in physiologically relevant [Ca2+]. The neutralization of residues known to be involved in high-affinity Ca2+ sensing (D362 and D367) prevented Ca2+-induced structural transitions in RCK1 but did not abolish Ca2+ binding. We provide evidence that the RCK1 domain is a high-affinity Ca2+ sensor that transduces Ca2+ binding into structural rearrangements, likely representing elementary steps in the Ca2+-dependent activation of human BKCa channels.

Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker

Trafficking of the pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels to the cell surface represents an important regulatory step in controlling BK channel function. Here, we identify multiple trafficking signals within the intracellular RCK1-RCK2 linker of the cytosolic C terminus of the channel that are required for efficient cell surface expression of the channel. In particular, an acidic cluster-like motif was essential for channel exit from the endoplasmic reticulum and subsequent cell surface expression. This motif could be transplanted onto a heterologous nonchannel protein to enhance cell surface expression by accelerating endoplasmic reticulum export. Importantly, we identified a human alternatively spliced BK channel variant, hSloΔ579–664, in which these trafficking signals are excluded because of in-frame exon skipping. The hSloΔ579–664 variant is expressed in multiple human tissues and cannot form functional channels at the cell surface even though it retains the putative RCK domains and downstream trafficking signals. Functionally, the hSloΔ579–664 variant acts as a dominant negative subunit to suppress cell surface expression of BK channels. Thus alternative splicing of the intracellular RCK1-RCK2 linker plays a critical role in determining cell surface expression of BK channels by controlling the inclusion/exclusion of multiple trafficking motifs.

Structure of the gating ring from the human large-conductance Ca(2+)-gated K(+) channel.

High-conductance Ca2+-gated K+ (BK) channels are essential for many biological processes such as smooth muscle contraction and neurotransmitter release1-4. This group of channels can be activated synergistically by both voltage and intracellular Ca2+, with the large C-terminal intracellular portion being responsible for Ca2+ sensing5-13. Here we present the crystal structure of the entire cytoplasmic region of the human BK channel in a Ca2+ free state. The structure reveals four intracellular subunits, each comprising two tandem RCK domains, assembled into a gating ring similar to that seen in the MthK channel14 and likely representing its physiological assembly. Three Ca2+ binding sites including the Ca2+ bowl are mapped onto the structure based on mutagenesis data. The Ca2+ bowl, located within the second RCK domain, forms an EF-hand like motif and is strategically positioned close to the assembly interface between two subunits. The other two Ca2+ (or Mg2+) binding sites, Asp367 and Glu374/Glu399, are located on the first RCK domain. The Asp367 site has high Ca2+ sensitivity and is positioned in the groove between the N- and C- terminal subdomains of RCK1, whereas the low affinity Mg2+-binding Glu374/Glu399 site is positioned on the upper plateau of the gating ring and close to the membrane. Our structure also contains the linker connecting the transmembrane and intracellular domains, allowing us to dock a voltage-gated K+ channel pore of known structure onto the gating ring with reasonable accuracy and generate a structural model for the full BK channel.

The RCK2 Domain Uses a Coordination Site Present in Kir Channels to Confer Sodium Sensitivity to Slo2.2 Channels

Slo2 Na(+)-activated potassium channels are widely expressed in neurons and other cells, such as kidney, heart, and skeletal muscle. Although their important physiological roles continue to be appreciated, molecular determinants responsible for sensing intracellular Na(+) remain unknown. Here we report identification of an Na(+) regulatory site, similar to an Na(+) coordination motif described in Kir channels, localized in the RCK2 domain of Slo2.2 channels. Molecular simulations of the homology-modeled Slo2.2 RCK2 domain provided structural insights into the organization of this Na(+) coordination site. Furthermore, free energy calculations reproduced the experimentally derived monovalent cation selectivity. Our results suggest that Slo2.2 and Kir channels share a similar mechanism to coordinate Na(+). The localization of an Na(+) sensor within the RCK2 domain of Slo2.2 further supports the role of RCK (regulators of conductance of K(+)) domains of Slo channels in coupling ion sensing to channel gating.

Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca(2+) and voltage-activated K(+) channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca(2+)-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K(d) = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K(d) = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.

Structure of the MthK RCK in complex with cadmium

RCK is a cytoplasmic regulatory domain of calcium-gated potassium channels. Binding of Ca(2+) by RCK leads to channel activation through a series of yet unknown conformational changes. Structures of the K(+) channel, MthK, and its cytoplasmic RCK domain revealed two binding sites for Ca(2+) per dimer. We determined the crystal structure of RCK in complex with Cd(2+) at 2.2A resolution. Cd(2+) activates MthK more efficiently, and binds at the same binding sites for Ca(2+) but with reduced coordination number. Two additional binding sites for Cd(2+) are found per dimer; one on the main Rossman-fold lobe, and the other on the small lobe of RCK. Using patch-clamp experiments, we demonstrate that Cd(2+) binding to these novel sites enhances activation by Cd(2+) and not by Ca(2+). The structure reveals a large negatively charged surface patch in the proximity of the Ca(2+)/Cd(2+) binding sites, charge neutralization of which appears to promote the channel open state.

Identification of Divalent Cation Coordinating Residues in a K+ Channel RCK Domain by NMR Spectroscopy

TvoK is a prokaryotic K+ channel whose gating is modulated by divalent cation-binding to a carboxy-terminal RCK domain. To gain insight toward mechanisms underlying divalent cation binding and subsequent conformational changes, we measured chemical shift perturbations upon ligand binding in the soluble cytoplasmic RCK domain of TvoK using heteronuclear NMR spectroscopy.Uniformly 15N-labeled, highly deuterated TvoK RCK domain was overexpressed in E.coli and purified by affinity and gel-filtration chromatography. 15N-HSQC spectra showed well-dispersed crosspeaks corresponding to >85% of the 238 predicted backbone NH groups. Five-point titration experiments using 0 to 100 ∈1/4M Mn2+ identified 12 residues that surrounded a putative divalent cation binding site, as indicated by spectral line-broadening due to the paramagnetic relaxation enhancement effect of Mn2+ (Mn-PRE). Partial resonance assignments, made through a combination of HNCA experiments and residue-specific 15N-labeling, identify D192 and E226 as key residues in divalent cation coordination, as indicated by high sensitivity to Mn-PRE (Kapp< 10 ∈1/4M). Further resonance assignments will identify remaining residues that lie within ∼15 A of the binding site. These experiments may reveal differences between the structural and chemical properties of the TvoK binding site and the Ca2+-selective binding site of the MthK RCK domain, which may underlie differential selectivities of MthK and TvoK RCK domains for divalent cations.

The Na+-Activated Potassium Channel Slack Shares a Similar Na+ Coordination Site with Kir3 Channels

Characteristics of Na+ activated potassium channel (Slack or Slo2.2) currents, including high conductance, rundown, regulation by Na+, Cl- and phosphorylation have long been reported but underlying mechanisms remain unknown. Here we report identification of a sodium regulatory site in the RCK2 domain of Slack channels by screening the C-terminus with the conserved sodium coordination motif of Kir channels. While the charge preserving D818E mutation exhibited similar Na+ sensitivity as the wild-type Slack channel, both a neutralization mutant D818N and a charge reversal D818R mutant within this site dramatically decreased Na+ sensitivity. Thus, D818 is engaged in an important electrostatic interaction with Na+. Similarly, the H823N mutant within this site also greatly decreased Na+ sensitivity of Slack channels. Simulations of the Slack RCK2 domain based on the crystallized structure of a prokaryotic RCK domain structure (Jiang et al., 2001, Neuron 29:593) provided a model of the Na+ coordination site in Slack channels. Moreover, simulations of the Na+ coordination site in Slo2.2 channels predicted a 5∼7 fold selectivity for Na+ over Li+ that were confirmed by electrophysiological data. Our results suggest that the Slack channel shares a similar Na+ regulatory mechanism with Kir channels but with important differences, such as an intricate coupling mechanism to Cl- co-regulation and possibly additional Na+ sensitive sites.

Prediction of Calcium Binding Site in the RCK1 Domain of BKCa Channel Using Multisite Cation Model

Calcium plays a major role in controlling the opening and closing of the large conductance BKCa channels. Two high affinity binding sites have been identified in the channel structure and one of these sites is the DRDD loop in the N-terminus of the RCK1 domain. Mutation of the first aspartate in this conserved DRDD motif significantly reduces Ca2+ sensitivity and hence this residue has been implicated as a coordinating group in the binding site. Here we present results on the prediction of the Ca2+ binding site based on a series of detailed computational studies. We use a novel multisite cation model for calcium ion to accurately simulate the ion-coordination. The basic protocol involves multiple iterations of random ion placement, implicit solvent molecular dynamics simulations and statistical analysis. Our resulting model matches very well with existing mutagenesis data, and subsequent explicit solvent molecular dynamics simulations have been performed using this Ca2+ bound structure. Comparison of the dynamics and conformations of the Ca2+ bound and unbound simulations reveal a concerted conformational change in the structure and suggest a potential mechanism for calcium dependent activation of these channels.

Voltage Sensor Activation Facilitates Magnesium-Gated Channel Opening in BK Channels

Under physiological conditions, Mg2+ is an intracellular activator of Ca2+- and voltage-activated potassium (BK) channels. To investigate gating by Mg2+ acting through its low affinity site located under each S4 voltage sensor (Yang et al. 2007, 2008; Horrigan & Ma 2008), we studied a BK channel in which the high affinity Ca2+ sites in both the RCK1 domain and the calcium bowl were disabled by mutation. Using single-channel recording from inside-out patches to measure channel activation after voltage steps from −100 mV to +100 mV, we found that 10 mM Mg2+ reduces the latency to first opening after the voltage step and increases channel activation through an increase in the number of openings per burst and mean open duration. This suggests that Mg2+ can bind to both closed and open states of the channel, but it is not clear whether the closed-state binding occurs at the negative or positive potential. Therefore we recorded single-channel activity in macro-patches held at a constant −50 or −100 mV. At −50 mV, when the voltage sensors are occasionally activated, 10 mM Mg2+ decreases the duration of the closed intervals between bursts of activity and increases burst duration through an increase in the number of openings per burst and mean open duration. At −100 mV, when the voltages sensors are mainly deactivated, 10 mM Mg2+ has little effect on the closed intervals between bursts or mean open duration, and most of the bursts are unitary, consisting of a single brief opening. The above data are consistent with a model in which Mg2+ can bind to the BK channel in the closed conformation when the voltage sensors are activated. The bound Mg2+ then facilitates opening. Supported by NIH grant AR32805.